Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation
Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation
复制标题
通过原核移植对 CRISPR-Cas9 进行时空控制，进行精确的等位基因特异性基因组编辑
DOI：
10.1038/s41467-020-18391-y
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发表时间：
2020-09-14
影响因子：
16.6
通讯作者：
Gao, Shaorong
中科院分区：
文献类型：
--
作者：
Li, Yanhe;Weng, Yuteng;Gao, Shaorong
通过将Cas9和sgRNA注射到受精卵中产生的基因靶向动物模型通常伴随着不期望的双链断裂（DSB）诱导的副产物和由于不可控的Cas9靶向活性而导致的随机双等位基因靶向。在这里，我们建立了一种亲本等位基因特异性基因靶向（Past-CRISPR）方法，基于原核转移介导的细胞质稀释可以有效终止Cas9活性的详细观察。我们在胚胎中应用这种方法以有效地靶向感兴趣基因的给定亲本等位基因，并且由于Cas9活性的时空控制而观察到很少的基因组嵌合现象。这种方法使我们能够快速探索个体亲本效应的功能，并构建具有单一基因组变化的动物模型。更重要的是，Past-CRISPR还可以用于治疗应用或疾病模型构建。
Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.